Preparation containing acetovanillone and paeonol

Abstract

The present invention relates to a composition comprising 2-hydroxy-4-methoxyacetophenone (paeonol) or an isomer thereof and 4-hydroxy-3-methoxyacetophenone (acetovanillone) or an isomer thereof for use in the treatment of senescence (e.g., by modulating the mechanism of action through senescence phenotype).

Description

[0001] The present invention relates to a composition comprising 2-hydroxy-4-methoxyacetophenone (paeonol) or an isomer thereof and 4-hydroxy-3-methoxyacetophenone (apocynin) or an isomer thereof, for use in treating aging (e.g., through a senomorphic mechanism of action).

[0002] Cellular senescence is important for maintaining tissue homeostasis. As a general term, senescence refers to biological aging. Dysregulation of senescence is associated with many human diseases and the aging process, and is a major risk factor for debilitating conditions, including cancer, cardiovascular disease, and neurodegenerative diseases.

[0003] Cellular senescence is a mechanism that permanently arrests cell growth. Senescence can be induced by stresses such as telomere erosion, mitochondrial degeneration, DNA damage, and oxidative stress caused by repeated cell division.

[0004] Since cellular senescence is characterized by the cessation of cell division, it ensures that aged or damaged cells cannot continue their genome. This can prevent carcinogenesis in normal development, however, senescent cells accumulate and produce pro-inflammatory and inflammatory cytokines, chemokines and enzymes, collectively known as the senescence-associated secretory phenotype (SASP) factors. SASP factors are believed to disrupt tissue structure and function and play a significant role in the development of age-related diseases. These diseases include, but are not limited to, osteoporosis; sarcopenia; lung diseases such as chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), pulmonary hypertension (PH), and pulmonary arterial hypertension (PAH); liver diseases such as acute liver injury, chronic liver disease, and hepatic steatosis; kidney diseases such as acute kidney injury (AKI), chronic kidney disease (CKD), tubulointerstitial fibrosis (TIF), glomerulosclerosis, and vascular sclerosis; vascular diseases such as atherosclerosis; type 1 diabetes; age-related macular degeneration; and neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease, frontotemporal dementia, amyotrophic lateral sclerosis, and multiple sclerosis.

[0005] Osteoporosis is an age-related skeletal disease characterized by low bone mass and gradual degeneration of bone tissue, leading to an increased risk of fractures. It is associated with the accumulation of senescent cells in the skeleton (osteocytes). Excessive production of proinflammatory cytokines by senescent osteocytes promotes chronic inflammation and forms a toxic microenvironment, which leads to age-related bone loss. Clearance of senescent cells and / or SASP is expected to slow or prevent age-related osteoporosis.

[0006] Sarcopenia is an age-related musculoskeletal disorder involving a progressive loss of muscle mass, strength, and function. Senescence of muscle stem cells is thought to lead to a loss of muscle regenerative potential, while elevated levels of SASP factors secreted by senescent cells lead to chronic inflammation, which may accelerate protein degradation and myofiber loss, leading to sarcopenia. Clearance of senescent cells and / or the SASP has the potential to treat sarcopenia.

[0007] Several lung diseases are associated with aging, particularly chronic obstructive pulmonary disease (COPD), an inflammatory lung disease that gradually limits airflow to the lungs; idiopathic pulmonary fibrosis (IPF), a chronic, fatal lung disease in which scarring of the tissue surrounding the alveoli in the lungs occurs; and pulmonary hypertension (PH) and pulmonary arterial hypertension (PAH). The accumulation of senescent fibroblasts and lung epithelial cells, as well as the SASP, are closely associated with lung disease pathology. Clearance of senescent cells and / or the SASP may have potential as a therapeutic strategy for treating COPD, IPF, PH, and PAH.

[0008] Liver conditions are also associated with aging, particularly acute liver injury, chronic liver disease, and hepatic steatosis (fat accumulation in the liver unrelated to alcohol consumption). A close correlation has been found between senescence markers in hepatocytes and acute liver injury, chronic liver disease, hepatic fat accumulation, and steatosis. Therefore, clearance of senescent cells and / or the SASP may have the potential to treat liver conditions.

[0009] Kidney diseases (especially acute kidney injury (AKI), chronic kidney disease (CKD), tubulointerstitial fibrosis (TIF), glomerulosclerosis, and vascular sclerosis) are closely associated with cellular senescence. Clearance of senescent cells and / or SASP may become a new treatment for kidney diseases.

[0010] Atherosclerosis is a chronic arterial disease characterized by excessive deposition of fatty material (plaque or atheroma) on the inner walls of arteries. Senescence of lipid-laden macrophages ("foam cells") has been shown to be detrimental at all stages of atherosclerosis. Accumulation of senescent foam cells in the subendothelial space at the onset of atherosclerosis is believed to promote atheroma formation and maturation via the SASP. Therefore, clearance of senescent cells and / or the SASP may have therapeutic potential for atherosclerosis.

[0011] Type 1 diabetes (T1D) is an autoimmune disease characterized by the progressive loss of pancreatic beta cells, leading to hyperglycemia. The pathogenesis of T1D is closely linked to beta cell senescence. Targeted elimination of senescent beta cells has been shown to prevent T1D and may be a viable approach for T1D treatment.

[0012] Age-related macular degeneration (AMD) is an age-related condition in the macula of the eye that affects central vision. Aging of retinal and choroidal tissue cells has been shown to be a significant factor in the onset and development of AMD. Removal of senescent cells and / or SASP may have the potential to treat AMD.

[0013] Senescence of astrocytes, microglia, oligodendrocytes, neurons, and neural stem cells has been implicated in the development of neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, frontotemporal dementia, amyotrophic lateral sclerosis, and multiple sclerosis.

[0014] Various studies in mice and humans suggest that targeting senescent cells and / or the SASP may be an effective strategy to delay age-related pathologies, increase healthspan, and potentially extend lifespan.

[0015] Anti-aging therapeutic compounds can target senescence, and these compounds are divided into two categories: senolytic compounds and senomorphic compounds. If the administration of a compound results in the selective elimination of senescent cells and thus leads to a reduction in both the number of senescent cells and the total number of cells, the compound is referred to as a senolytic compound. A senomorphic compound is a compound whose administration results in the selective inhibition of the SASP and the maintenance or increase of the total number of cells.

[0016] Given the key roles of senescent cells and the SASP as risk factors for age-related pathologies, there is a need for a composition for treating aging.

[0017] According to the present invention, there is provided a composition comprising 2-hydroxy-4-methoxyacetophenone (paeonol) or an isomer thereof and 4-hydroxy-3-methoxyacetophenone (vanilla acetophenone) or an isomer thereof, for use in treating aging, wherein administration of the composition results in a decrease in the number of senescent cells and a simultaneous increase in the number of total viable cells. In this context, the term "viable" means cells that are alive and capable of reproduction. Cell viability can be assessed by staining with a viability dye (such as trypan blue or calcein-AM) or by staining with Hoechst dye, but counting intact cell nuclei. The composition according to the present invention can exert its effect through a senescent cell clearance mechanism or a senescence phenotype regulation mechanism. Preferably, the composition according to the present invention exerts its effect through a senescence phenotype regulation mechanism. Preferably, the composition according to the present invention exerts its effect through a senescence phenotype regulation mechanism, which leads to selective inhibition of the SASP.

[0018] According to the present invention, in another aspect, a composition is provided, comprising paeonol or an isomer thereof and vanillyl acetonide or an isomer thereof, and the composition is used to treat aging through an aging phenotype regulating mechanism.

[0019] According to the present invention, in another aspect, a composition is provided, comprising paeonol or an isomer thereof and vanillyl acetonide or an isomer thereof, and the composition is used for treating aging through a senescent cell clearance mechanism.

[0020] Paeonol is 2-hydroxy-4-methoxyacetophenone and is shown in the following formula:

[0021]

[0022] Paeonol can be found in plant materials and plant extracts. For example, paeonol can be found in Paeonia suffruticosa, Paeonia lactiflora, Paeonia veitchii, Paeonia obovata, Rheum palmatum (rhizome), and Scutellaria baicalensis (root). The compositions (and medicaments) of the present invention comprise 2-hydroxy-4-methoxyacetophenone (paeonol).

[0023] According to our research, paeonol is a solid with a low melting point of about 49.7°C.

[0024] Preferably, paeonol has been synthesized or extracted and purified from plants. This can be referred to as isolated paeonol. The amounts and ratios described herein refer to isolated paeonol. Less preferably, paeonol can be present in the compositions (and medicaments) according to the present invention as a direct extract from a plant (i.e., as part of an unresolved mixture of compounds in the form of an unpurified plant or root extract). These will be referred to as "natural form" paeonol or "natural paeonol." For example, paeonol present in the compositions (and medicaments) according to the present invention in the form of a peony will be referred to as "natural paeonol." The term "natural form" paeonol or "natural paeonol" includes glycosides of paeonol, such as those found in the plant species in which paeonol is found. Such glycosides include, for example, paeonin, paeonolide, and paeonoside. If natural paeonol is used, the skilled person will readily understand how to adjust the ratio to provide a formulation of the present invention.

[0025] Compared with natural paeonol, isolated paeonol provides more consistent paeonol quality. Isolated paeonol can also be produced on a larger scale than natural paeonol. Therefore, isolated paeonol is preferably used in this application.

[0026] Vanilla acetophenone is the plant phenol 4-hydroxy-3-methoxyacetophenone and has the following formula:

[0027]

[0028] According to our research, vanillone is a solid with a higher melting point of about 114.6°C.

[0029] Vanilla acetone is found in plant matter and plant extracts, for example in extracts of Picrorrhiza kurroa, Apocynum cannabinium, Apocynum venatum, Apocynum androsaemifolium, and vanilla species such as Vanilla planifolia.

[0030] The compositions (and medicaments) of the present invention comprise 4-hydroxy-3-methoxyacetophenone (vanillic acetone). Preferably, vanillic acetone has been synthesized or extracted and purified from a plant. This can be referred to as isolated vanillic acetone. The amounts and ratios described herein refer to isolated vanillic acetone.

[0031] Vanilla acetone may be present in the compositions (and medicaments) according to the invention as a direct extract from plants (such as those mentioned above) (e.g., in the form of unpurified plant or root extracts as part of an unresolved mixture of compounds). These will be referred to as "natural form" of vanilla acetone or "natural vanilla acetone." For example, vanilla acetone present in the compositions (and medicaments) according to the invention in the form of Picrorhizon will be referred to as "natural vanilla acetone." The term "natural vanilla acetone" or "natural form" of vanilla acetone also includes glycosides of vanilla acetone, such as those found in the plant species in which vanilla acetone is found. Such glycosides include, for example, androsin and other iridoid glycosides.

[0032] The composition may contain vanillone in the form of an unpurified plant or root extract as part of an unresolved mixture of compounds: "natural" vanillone. Picrorhizon is a standardized form based on a standardized iridoid glycoside fraction; such forms are well known. Standardized forms of Picrorhizon include Picrorhizon standardized to "Kutkin minimum 4%." Picrorhizon is obtained by crystallization and consists of the glycosides picroside I and kutoside in a 1:2 ratio, along with other minor glycosides (Sing and Rastogi, 1972; Ansari et al., 1988).

[0033] Compared with natural vanilla acetone, isolated vanilla acetone provides more consistent vanilla acetone quality. Isolated vanilla acetone can also be produced on a larger scale than natural vanilla acetone. Therefore, isolated vanilla acetone is preferably used in the present invention.

[0034] As mentioned above, the composition may include vanillone in its natural form, although this is less preferred. If natural vanillone is used, a skilled artisan will readily understand how to adjust the ratios to produce the formulations of the present invention. However, if this is the case, it may be necessary to limit the amount of Picrorhizon to prevent side effects (such as gastric discomfort that may occur due to other phytochemicals in Picrorhizon). However, it should be noted that most human subjects can consume up to 2,000 mg of Picrorhizon (minimum 2% picrocerin) per day without discomfort.

[0035] Isomers of paeonol and vanillone are known in the art. Isomers of paeonol and vanillone include 2-hydroxy-3-methoxyacetophenone "orthoacetovanillone" (CAS: 703-98-0), 2-hydroxy-5-methoxyacetophenone (CAS: 705-15-7), 3-hydroxy-4-methoxyacetophenone "isoacetovanillone" (CAS: 6100-74-9), and 4-hydroxy-2-methoxyacetophenone "isopaeonol" (CAS: 493-33-4).

[0036] Paeonol and vanillic acetone are isomers of each other. It will be understood that according to the present invention, a composition must comprise two different active compounds (e.g., paeonol itself and vanillic acetone itself). Preferably, a composition according to the present invention comprises paeonol itself and vanillic acetone itself. In this article, the name "APPA" is used to represent a composition comprising paeonol and vanillic acetone, as described above.

[0037] Applicants have discovered that compositions of the present invention (eg, APPA) have the ability to reduce the number of senescent cells in a human chondrocyte cell line.

[0038] The applicant further found that the administration of the composition of the present invention (eg, APPA) to a human chondrocyte cell line resulted in an increase in the total cell number. Therefore, the composition of the present invention (eg, APPA) exhibited a senescent phenotype regulatory effect on a human chondrocyte cell line (see Examples).

[0039] The use of paeonol or its isomers and vanillyl acetonide or its isomers as drugs for clearing senescent cells and / or regulating senescent phenotypes can be regarded as a significant advancement, which opens up new therapeutic avenues for clearing or delaying the adverse effects of cellular senescence and the resulting age-related pathologies.

[0040] The composition according to the invention may have a ratio (by weight) of paeonol or its isomers to vanillyl ethyl ketone or its isomers of 3:2 to 9:1. For example, the composition according to the invention may have a ratio (by weight) of paeonol or its isomers to vanillyl ethyl ketone or its isomers of 3:2 to 5:1. For example, the composition according to the invention may have a ratio (by weight) of paeonol or its isomers to vanillyl ethyl ketone or its isomers of 5:1 to 9:1.

[0041] The composition according to the present invention can be a liquid preparation. The composition according to the present invention can be a liquid preparation, and the liquid preparation is made of a eutectic mixture comprising the active compound paeonol or its isomer and vanillyl acetone or its isomer, and the liquid preparation also comprises at least one excipient. The excipient can be a diol or a diol derivative, a polyol or its ester and / or ether. In one embodiment, the preparation has a weight ratio of the total active compound to the excipient of 2:3 to 19:1. Preferably, the weight ratio of the total active compound to the excipient is 2:3 to 9:1. Preferably, the weight ratio of the total active compound to the excipient is 64:36. Preferably, the preparation is a stable liquid at room temperature (15 to 25°C).

[0042] The composition according to the present invention may be a solid preparation such as a powder, tablet or pill.

[0043] The composition according to the present invention can be used to treat age-related diseases.

[0044] The composition according to the present invention can be used to treat osteoporosis. The composition according to the present invention can be used to treat sarcopenia. The composition according to the present invention can be used to treat chronic obstructive pulmonary disease (COPD). The composition according to the present invention can be used to treat idiopathic pulmonary fibrosis (IPF). The composition according to the present invention can be used to treat lung diseases, such as pulmonary hypertension (PH) and pulmonary arterial hypertension (PAH). The composition according to the present invention can be used to treat liver diseases, such as acute liver injury, chronic liver disease and hepatic steatosis. The composition according to the present invention can be used to treat kidney diseases, such as acute kidney injury (AKI), chronic kidney disease (CKD), tubulointerstitial fibrosis (TIF), glomerulosclerosis and vascular sclerosis. The composition according to the present invention can be used to treat vascular diseases, such as atherosclerosis. The composition according to the present invention can be used to treat type 1 diabetes. The composition according to the present invention can be used to treat age-related macular degeneration (AMD). The composition according to the present invention can be used to treat Alzheimer's disease (AD), Parkinson's disease, frontotemporal dementia, amyotrophic lateral sclerosis or multiple sclerosis.

[0045] The composition according to the invention may be administered orally.

[0046] Pharmaceutical composition of the present invention can be mixed with well-known composition, it is used for any drug administration route, for example oral, rectal, parenteral, transdermal (for example patch technology or transdermal gel preparation), intravenous, intramuscular, subcutaneous, intracisternal, intravaginal, intraperitoneal, local (powder, ointment or drops), as oral spray or nasal spray.Typical composition comprises pharmaceutical carrier, such as aqueous solution, nontoxic excipient (comprising salt and preservative), buffer agent etc., as described in Lei Mingdun's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences) the 15th edition (Matt Publishing Company, 1975) 1405 to 1412 pages and 1461 to 1487 pages and National Formula XIV the 14th edition (American Pharmaceutical Association, 1975) etc.

[0047] Examples of suitable aqueous and nonaqueous pharmaceutical carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate.

[0048] The compositions of the present invention may also contain additives such as, but not limited to, preservatives, wetting agents, emulsifiers, and dispersants. Antibacterial and antifungal agents may also be included to prevent the growth of microorganisms, and include, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. In addition, it is desirable to include isotonic agents such as sugars, sodium chloride, and the like.

[0049] According to the present invention, in another aspect, provided is the use of vanillone or its isomers and paeonol or its isomers in preparing a medicament for treating aging.

[0050] According to the present invention, in another aspect, provided is the use of vanillone or its isomers and paeonol or its isomers in the preparation of a medicament for treating age-related diseases.

[0051] According to the present invention, in another aspect, there is provided a use of vanillone or its isomers and paeonol or its isomers in the preparation of a medicament for treating osteoporosis; sarcopenia; lung diseases such as chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), pulmonary hypertension (PH) and pulmonary arterial hypertension (PAH); liver diseases such as acute liver injury, chronic liver disease and hepatic steatosis; kidney diseases such as acute kidney injury (AKI), chronic kidney disease (CKD), tubulointerstitial fibrosis (TIF), glomerulosclerosis and vascular sclerosis; vascular diseases such as atherosclerosis; type 1 diabetes; age-related macular degeneration (AMD); and neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease, frontotemporal dementia, amyotrophic lateral sclerosis and multiple sclerosis.

[0052] According to the present invention, in another aspect, provided is the use of vanillone or its isomers and paeonol or its isomers in the preparation of a medicament for treating Alzheimer's disease, Parkinson's disease, frontotemporal dementia, amyotrophic lateral sclerosis or multiple sclerosis.

[0053] According to the present invention, a method for treating aging is provided, comprising the following steps: administering a composition comprising vanillone or an isomer thereof and paeonol or an isomer thereof to a patient in need thereof.

[0054] According to the present invention, a method for treating age-related diseases is provided, comprising the following steps: administering a composition comprising vanillone or an isomer thereof and paeonol or an isomer thereof to a patient in need thereof.

[0055] According to the present invention, in another aspect, a method for treating the following diseases is provided: osteoporosis, sarcopenia, lung diseases such as chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), pulmonary hypertension (PH) and pulmonary arterial hypertension (PAH), liver diseases such as acute liver injury, chronic liver disease and hepatic steatosis, kidney diseases such as acute kidney injury (AKI), chronic kidney disease (CKD), tubulointerstitial fibrosis (TIF), glomerulosclerosis and vascular sclerosis, vascular diseases such as atherosclerosis, type 1 diabetes, age-related macular degeneration (AMD), Alzheimer's disease (AD), Parkinson's disease, frontotemporal dementia, amyotrophic lateral sclerosis or multiple sclerosis; the method comprises the following steps: administering a composition to a patient in need thereof, the composition comprising vanillone or an isomer thereof and paeonol or an isomer thereof.

[0056] The present invention will now be described in detail with reference to the following examples and accompanying drawings, in which:

[0057] Example 1 demonstrates the aging phenotype-regulating activity of APPA in the chondrocyte cell line TC28a2;

[0058] Throughout the examples, '*' indicates 'compared to the results of basal measurements'; '#' indicates 'compared to the results of etoposide alone measurements';

[0059] Figure 1 Shown are the effects of APPA on the levels of the senescence-associated secretory phenotype (SASP) marker β-galactosidase in TC28a2 chondrocytes when induced by exposure to IL-6 or etoposide (Eto);

[0060] Figure 2 Shown are the effects of APPA, AP, and PA on chondrocyte viability in human TC28a2 chondrocytes (using DRAQ7);

[0061] Figure 3 Shown is the effect of APPA on the number of viable TC28a2 cells exposed to etoposide (Eto);

[0062] FIG4 shows the effects of APPA and early and late TC28a2 apoptosis in TC28a2 cells exposed to IL-6 or etoposide (Eto);

[0063] Figure 4a :Effects of APPA, AP and PA on early apoptosis in human TC28a2 chondrocytes;

[0064] Figure 4b :Effects of APPA, AP and PA on late apoptosis in human TC28a2 chondrocytes;

[0065] Figure 5 Shown are the effects of APPA and its components AP and PA on the levels of the senescence-associated secretory phenotype (SASP) marker β-galactosidase in TC28a2 chondrocytes when induced by exposure to etoposide;

[0066] Example 2 demonstrates the aging phenotype regulatory activity of APPA in primary human chondrocytes;

[0067] FIG6 shows the effect of APPA on SA-β-Gal activity and CDKN1A gene expression levels in primary human chondrocytes exposed to etoposide and OSM;

[0068] Figure 6a :Effects of different concentrations of etoposide (+OSM) on the level of β-galactosidase, a marker of the senescence-associated secretory phenotype (SASP);

[0069] Figure 6b :Effects of different concentrations of etoposide (+OSM) on CDKN1A gene expression in primary human chondrocytes;

[0070] Figure 6c : Effect of APPA on the elevated levels of β-galactosidase, a marker of the senescence-associated secretory phenotype (SASP), induced by 20 μM etoposide (Eto) plus 10 ng / ml oncostatin M (OSM); “+” indicates “presence”; “-” indicates “absence”

[0071] Figure 6d : Effect of APPA on the expression of CDKN1A-related genes induced by 20 μM etoposide (Eto) plus 10 ng / ml oncostatin M (OSM); "+" indicates "presence"; "-" indicates "absence"

[0072] FIG7 shows the effects of APPA, AP, and PA on SA-β-Gal activity, CDKN1A gene expression level, BECN-1 gene expression level, and BCL2L13 expression level in primary human chondrocytes exposed to etoposide and OSM;

[0073] Figure 7a :The effect of 20 μM etoposide (Eto) plus 10 ng / ml oncostatin M (OSM) on the level of β-galactosidase, a marker of the senescence-associated secretory phenotype (SASP);

[0074] Figure 7b : Effects of APPA and its components on the elevated levels of β-galactosidase, a marker of the senescence-associated secretory phenotype (SASP), induced by 20 μM etoposide (Eto) plus 10 ng / ml oncostatin M (OSM);

[0075] Figure 7c : Effects of APPA and its components on the increase in CDNK1A gene expression induced by 20 μM etoposide (Eto) plus 10 ng / ml oncostatin M (OSM);

[0076] Figure 7d : Effects of APPA and its components on the increased gene expression of BECN-1 induced by 20 μM etoposide (Eto) plus 10 ng / ml oncostatin M (OSM);

[0077] Figure 7e : Effects of APPA and its components on the increased gene expression of BCL2LK13 induced by 20 μM etoposide (Eto) plus 10 ng / ml oncostatin M (OSM);

[0078] Drug preparation

[0079] APPA was provided in prepared vials (AP:PA ratio of 2:7), while AP and PA were obtained from Sigma (Sigma-Aldrich, St Louis, MO, USA). All compounds were dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich) at a final working concentration of 1 gr / ml and serially diluted.

[0080] Chondrocyte isolation

[0081] Written informed consent was obtained from all subjects and approval was obtained from the local ethics committee of the Galician Health Administration (CEIC).All procedures were performed in accordance with the principles set forth in the 1975 Helsinki Declaration (revised 2000).

[0082] Chondrocytes from human OA hip articular cartilage were isolated from 18 total hip replacement patients (5 male and 13 female patients, with a mean ± SD age of 86 ± 7.17 years and 79.71 ± 13.56 years, respectively) as described in (Maneiro E, Martín MA, de Andres MC, López-Armada MJ, Fernández-Sueiro JL, del Hoyo P, et al. Mitochondrial respiratory activity is altered inosteoarthritic human articular chondrocytes. Arthritis and rheumatism. 2003; 48(3): 700-8)).

[0083] T / C28a2 chondrocytes

[0084] In some experiments, the immortalized human juvenile chondrocyte cell line T / C28a2 (25) was used.

[0085] Human articular chondrocytes and T / C28a2 cell lines were equilibrated overnight at 37°C in a humidified 5% CO2 atmosphere in Dulbecco's modified Eagle's medium (DMEM) (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U / ml) and streptomycin (100 μg / ml) (Gibco).

[0086] Cell culture chondrocytes and T / C28a2 cell lines:

[0087] The cells were plated at 5 × 10 4 Cells were plated at a density of 10 cells / well. Multiwell 96-well plates (MW96) (Corning, NY, USA) or 1.8 × 10 5 Cells were plated at a density of 10 cells / well in MW12 plates (Corning, NY, USA) for analysis. Cells were equilibrated overnight in DMEM medium containing 5% or 2% FBS at 37° C. in 5% CO 2 . Cells were stimulated with etoposide (20 μM for chondrocytes or 5 μM in the case of cell lines) (Sigma-Aldrich). Example

[0088] Example 1: Demonstration of senescence phenotype regulatory activity in the chondrocyte cell line TC28a2

[0089] The applicants found that APPA can reduce the level of the SASP marker β-galactosidase in the human chondrocyte cell line TC28a2 – indicating a decrease in the number of senescent cells – but increase the total number of cells. Therefore, APPA shows a regulatory effect on the senescent phenotype of the human chondrocyte cell line TC28a2.

[0090] A widely used marker of senescence is increased levels of senescence-associated β-galactosidase (SA-β-Gal) activity (visualized by quantification of fluorescein di-β-D-galactopyranoside (FDG) using flow cytometry).

[0091] Therefore, the effect of APPA on the number of senescent cells was evaluated by quantifying β-galactosidase activity using flow cytometry. β-galactosidase activity was detected by flow cytometry using fluorescein di-β-D-galactopyranoside (FDG; Thermo Fisher, Waltham, MA, USA). Non-fluorescent FDG is sequentially hydrolyzed by β-galactosidase, first to fluorescein monogalactoside and then to the highly fluorescent fluorescein. Enzyme-mediated hydrolysis of FDG can lead to enhanced fluorescence.

[0092] Cell cultures were pretreated with 5 μM etoposide or 20 ng / mL interleukin 6 (IL-6) for 72 hours (Sigma-Aldrich Merck KGaA, Darmstadt, Germany) to induce DNA damage as genotoxic stress, thereby causing cellular senescence; they were then pretreated with 10 nM bafilomycin A (Sigma-Aldrich) for 1 hour to regulate the intracellular pH. These conditions were evaluated with and without 10 μg / ml APPA for 24 hours. Fluorescein di-β-D-galactopyranoside (10 μM) was then added to the pretreatment medium. At the end of the incubation period, the cultures were washed with PBS, resuspended by trypsinization, and immediately analyzed using a FACScalibour flow cytometer (Becton Dickinson). Data were collected and analyzed using Cellquest software (Becton Dickinson). Each fluorescein signal was measured on an FL1 detector, and the median fluorescence intensity (arbitrary units) of the cell population was used to estimate β-galactosidase activity.

[0093] Figure 1 and Table 1 show the median fluorescence of FDG (median ± SEM) of human TC28a2 chondrocytes treated with APPA and compared with aged controls (20 ng / ml IL-6 or 5 μM etoposide). Figure 1 It was shown that APPA reduced the number of senescent cells, as indicated by decreased SA-β-Gal activity, in both TC28a2 cells exposed to IL-6 and TC28a2 cells exposed to etoposide.

[0094]

[0095] Table 1: Effects of APPA on the levels of senescence-associated β-galactosidase (SA-β-Gal) in human TC28a2 chondrocytes; * = relative to basal; # = relative to Eto.

[0096] Figure 2The effects of APPA, AP, and PA on chondrocyte viability (using DRAQ7) are shown.

[0097] A novel cell viability assay uses DRAQ7, a marker for apoptosis, necrosis, and dead cells. DRAQ7 stains the nuclei of dead and permeabilized cells but not the nuclei of intact, live cells.

[0098] To determine the effects of APPA (10 μg / ml), AP (2.3 and 10 μg / ml), and PA (7.7 and 10 μg / ml) on chondrocyte viability, a DRAQ7-based TM (Thermo Fisher Scientific). TM A dye that can be used to visualize apoptotic, necrotic, and dead cells, and the assay includes staining of nuclei in dead and permeabilized cells, but not intact live cells.

[0099] For the assay, cells were grown in flasks due to the large number of cells required. After 24 hours of different treatments, cells were trypsinized and centrifuged at 1500 rpm for 10 minutes (min). Harvested cells were suspended in 3 μM of the fluorescent dye DRAQ7. TM (Thermo Fisher) and incubated in the dark at room temperature (rt) for 10 minutes. The samples were run on a CytoFLeX flow cytometer (Beckman Coulter Inc. Ca, USA). At least 1×10 4 cells. Data were analyzed using CyExpert V 2.5 software (Beckman Coulter Inc). Positive control cells were treated with 5 μM etoposide (Sigma-Aldrich), while negative control (basal) cells were grown in fresh DMEM with 5% FBS. To determine the positions of the DRAQ7- and DRAQ7+ gates, one of each negative and positive control was stained with DRAQ7 dye, while the other was stained with PBS alone (data not shown).

[0100] Figure 2The effects of APPA (10 μg / ml), AP (2.3 and 10 μg / ml), and PA (7.7 and 10 μg / ml) on the percentage of DRAQ7-positive (non-viable) cells are shown, which were used to estimate cell viability in the presence of APPA, AP, or PA. The percentage of non-viable cells never exceeded 5.05%; this value was obtained with 10 μg / ml AP. The DRAQ7 data show that APPA and its components AP and PA did not affect cell viability at the concentrations used, thus ruling out the possibility that the reduction in cell senescence was due to cytotoxicity.

[0101] Figure 3 Shown is the effect of APPA on the total viable cell number in the chondrocyte cell line TC28a2.

[0102] In culture 8×10 4 After the cells were grown and reached 70%-80% confluence, the cells were pretreated with 5 μM etoposide for 72 hours, and then the culture medium was changed to Dulbecco's modified Eagle's medium (DMEM) with 2% fetal bovine serum (FBS), and the cells were cultured under these conditions for 48 hours. Finally, APPA (10 μg / ml) was added for another 24 hours. After incubation, the cells were fixed in 4% paraformaldehyde (Sigma-Aldrich) for 10 minutes at room temperature and then incubated in 0.2% Tween 20 (Sigma-Aldrich) for 5 minutes. After this step, the cells were incubated with the nuclear dye 2'-(4-ethoxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5'-di-1H-benzimidazole trihydrochloride (Hoechst 33258) (Sigma-Aldrich) for 5 minutes. After washing with phosphate buffered saline (PB), coverslips were mounted on microscope slides using Prolong Gold Antifade Reagent Mountant (ThermoFisher Scientific). Fluorescence was visualized and photographed under an Olympus BX61 fluorescence microscope. All samples were analyzed in duplicate with 3-5 fields of view per well, and the mean and standard deviation were calculated.

[0103] The results are Figure 3Figure 4 shows a cell-free culture of TC28a2 cells. When TC28a2 cells were incubated with 5 μM etoposide and 10 μg / ml APPA, the number of cells present in the culture increased in a statistically significant manner. Therefore, APPA was confirmed to exert its effect through the mechanism of action of regulating the aging phenotype; that is, it reduced the level of the SASP marker SA-β-Gal (indicating a decrease in the number of senescent cells), but increased the total number of viable cells. The sensitivity of the cells to apoptosis was then analyzed (Figure 4). With or without 10 μg / ml APPA (for 24 hours), the cells were incubated for 72 hours in the presence of 2 μM etoposide or 20 ng / mL IL-6 (Sigma-Aldrich). The cells were harvested by trypsinization and resuspended in 1 × annexin binding buffer, followed by the addition of 5 μL of annexin V-fluorescein isothiocyanate (FITC) and 5 μL of propidium iodide (PI; ImmunoStep, Salamanca, Spain). After incubation for 15 minutes, cells were analyzed using a FACsCalibur flow cytometer (Becton Dickinson, NJ, USA). The number of cells (1×10 4 ). CellQuest software (Becton Dickinson) was used to analyze the data. Apoptosis was analyzed by counting cells stained with both Annexin V-FITC and PI. This allowed differentiation of intact cells (Annexin V-FITC and PI negative) from cells in an early apoptotic state (Annexin V-FITC positive and PI negative), cells in a late apoptotic state (Annexin V-FITC and PI positive), and cells in a necrotic state (Annexin V-FITC negative and PI positive). The results were expressed as a percentage of cells positive for each dye and represent the mean ± standard error of the mean (SEM) of three independent experiments.

[0104] FIG4 shows the effect of APPA on early ( Figure 4a ) and late ( Figure 4b ) apoptosis. APPA did not have any significant effect on early or late apoptosis. Although increased apoptosis would be an alternative way to reduce the level of SASP markers, this would be associated with a decrease in the number of viable cells. Figure 3 As shown, the fact that APPA increased the number of viable cells is consistent with the apoptosis data, indicating that the reduction in senescence does not occur as a result of increased apoptosis.

[0105] Figure 5 Table 2 shows the effects of AP, PA and APPA on TC28a2 cell senescence induced by etoposide, as determined by the levels of SA-β-Gal. Figure 1In the present study, SA-β-Gal levels were assessed by quantifying fluorescein di-β-D-galactopyranoside (FDG) using flow cytometry. When AP and PA were tested in parallel with APPA in TC28a2 cells exposed to etoposide, only APPA resulted in a significant reduction in SA-β-Gal activity, although PA 7.7 μg / ml approached significance (p=0.054). The effect of PA 10 μg / ml was not significant. APPA reduced the number of senescent TC28a2 cells to a greater extent (indicated by lower median FDG fluorescence) than its individual components, AP or PA. The effect of APPA at 10 μg / ml was greater than expected based on (i) the effect of 10 μg / ml AP or 10 μg / ml PA and (ii) the additive effect of 2.3 μg / ml AP and 7.7 μg / ml PA. This suggests a synergistic effect between AP and PA in the combination product.

[0106]

[0107] Table 2: Effects of AP, PA and APPA on the levels of senescence-associated β-galactosidase (SA-β-Gal) in human TC28a2 chondrocytes; * = relative to basal; # = relative to Eto.

[0108] Example 2 - Demonstration of senescence phenotype regulatory activity in primary human chondrocytes

[0109] To confirm the above data on the chondrocyte cell line TC28a2, the applicants used primary human chondrocytes to evaluate the ability of APPA to regulate the aging phenotype.

[0110] To determine the optimal concentration of etoposide in human primary chondrocytes, concentrations of 2, 5, and 20 μM etoposide were combined with 10 ng / ml OSM and incubated for 48 hours. Figure 6a and 6b The optimal etoposide concentration was evaluated to be 20 μM.

[0111] Figure 6c The effect of APPA on the number of senescent cells, as expressed by SA-β-Gal levels, is shown in Table 3. As in Example 1, SA-β-Gal levels were evaluated by quantifying fluorescein di-β-D-galactopyranoside (FDG) using flow cytometry.

[0112]

[0113] The data shown in the table correspond to mean ± SEM, N = 8

[0114] Table 3: Effect of APPA on the number of senescent cells as determined by SA-β-Gal levels in primary human chondrocytes; * = relative to basal; # = relative to Eto+OSM.

[0115] CDKN1 (p21) is a genetic marker of cellular senescence that encodes cyclin-dependent protein kinase inhibitor 1A, a regulator of cell cycle progression. The expression of this gene is strictly regulated by the tumor suppressor protein p53. Activated p53 increases the expression of p21, which promotes aging. Studies of OA have shown that cell cycle-related proteins play a role in its pathology. Since the increase in the expression of genes that inhibit proliferation leads to cell cycle arrest, the changes that occur in senescent cells can also lead to increased production of cytokines, growth factors, and matrix metalloproteinases (Loeser RF. Aging and osteoarthritis: the role of chondrocyte senescence and aging changes in the cartilage matrix. Osteoarthritis Cartilage. 2009 Aug; 17(8): 971-9).

[0116] Evidence of cellular senescence in tissues from elderly individuals can be obtained by examining the presence of markers of senescence, including histological staining for senescence-associated (SA) β-galactosidase (SA-β-Gal), SA heterochromatin, increased p53, p21, and p16, and decreased Wnt21 (Campisi J, d'Adda di Fagagna F. Cellular senescence: when bad things happen to good cells. Nat Rev Mol Cell Biol. 2007 Sep;8(9):729-40. doi:10.1038 / nrm2233. PMID:17667954).

[0117] Effect of APPA on the expression level of CDKN1 Figure 6d The relative mRNA expression levels of CDKN1 were determined by quantitative real-time (RT)-PCR in the presence of positive stimulation + / - 10 μg / ml APPA. Data were obtained from eight independent donors. Values ​​are expressed as mean ± SEM and analyzed using the Mann Whitney test (*p ≤ 0.05).

[0118] use RNA extraction was performed using Sigma-Aldrich (Sigma-Aldrich) according to the manufacturer's protocol. 0.5 μg of RNA was reverse transcribed into cDNA using SuperScript VILO (Thermofisher Scientific, Waltham, Massachusetts, USA) according to the manufacturer's instructions. RT-PCR was performed using TaqMan Universal Master Mix (Roche) on a LightCycler 480-II Instrument (Roche, Mannheim, Germany). Results were analyzed using Qbase+ version 2.5 software (Biogazelle, Ghent, Belgium). Gene expression was calculated relative to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

[0119]

[0120] The data shown in the table correspond to mean ± SEM, N = 8

[0121] Table 4: Effect of APPA on the expression level of CDKN1 in primary human chondrocytes; * = relative to substrate; # = relative to Eto+OSM.

[0122] In primary human chondrocytes stimulated with 20 μM etoposide and 10 ng / ml oncostatin M, APPA significantly reduced SA-β-galactosidase levels, indicating a reduction in the number of senescent cells. APPA also reduced the expression of CDKN1A (p21), a genetic marker of cellular senescence. This is consistent with the results for TC28a2 in Example 1.

[0123] FIG. 7 and Tables 5 and 6 show the effects of AP, PA, and APPA on the levels of SA-β-Gal and the gene expressions of CDKN1A (p21), BECN-1, and BCL2L13 in human chondrocytes.

[0124] Figure 7b The effects of AP, PA, and APPA on the number of senescent cells, as expressed by SA-β-Gal levels, are shown in Table 5. As in Example 1 and Figure 6, SA-β-Gal levels were evaluated by quantifying fluorescein di-β-D-galactopyranoside (FDG) using flow cytometry.

[0125]

[0126]

[0127] Table 5: Effects of APPA, AP and PA on the number of senescent cells as represented by SA-β-Gal levels in primary human chondrocytes; * = relative to basal; # = relative to Eto+OSM.

[0128] When AP, PA, and APPA were tested in human chondrocytes, only APPA significantly reduced SA-β-Gal levels; AP and PA alone had no significant effect (see Table 1). Figure 7b Based on the effects of (i) 10 μg / ml AP or 10 μg / ml PA and (ii) the additive effects of 2.3 μg / ml AP and 7.7 μg / ml PA, the effect of 10 μg / ml APPA was superior to that expected. This again suggests a synergistic effect between AP and PA in the combination product.

[0129] Effects of APPA, AP and PA on CDKN1A gene expression Figure 7c and are shown in Table 6. Relative mRNA expression levels of cyclin-dependent kinase inhibitor 1A (CDKN1) were determined by quantitative real-time (RT)-PCR in the same manner as described above. Data were obtained from six independent donors. Values ​​are expressed as mean ± SEM and analyzed using the Mann Whitney test (*p ≤ 0.05; #p ≤ 0.05). *Relative to basal conditions, #Relative to 20 μM etoposide + 10 ng / ml oncostatin M.

[0130] Mean ± SEM P base 0.7738±0.10 Eto 20μM+OSM 10ng / ml 1.893±0.48 0.0152* Eto 20μM+OSM 10ng / ml+APPA 10μg / ml 0.9049±0.07 0.026# Eto 20μM+OSM 10ng / ml+AP 2.3μg / ml 1.0102±0.25 Eto 20μM+OSM 10ng / ml+AP 10μg / ml 1.617±0.32 Eto 20μM+OSM 10ng / ml+PA 7.7μg / ml 1.451±0.18 Eto 20μM+OSM 10ng / ml+10μg / ml 0.8635±0.17 APPA 10 μg / ml 0.9009±0.24 0.0303#

[0131] Table 6: Effects of APPA, AP and PA on the expression level of CDKN1A (p21) in primary human chondrocytes; * = relative to basal; # = relative to Eto+OSM.

[0132] Figure 7c Table 6 shows that APPA 10 μg / ml significantly reduced the level of CDKN1A (p21) expression (1.893±0.48 vs. 0.904±0.07, p=0.05), as did PA 10 μg / ml (1.893±0.48 vs. 0.869±0.17, p=0.05), while neither concentration of AP nor PA 7.7 μg / ml had any significant effect.

[0133] Figure 7dThe effects of APPA, AP, and PA on the gene expression of BECN-1 are shown. BECN-1 is a regulator of autophagy; the encoded protein, Beclin 1, is a component of the phosphatidylinositol 3-kinase (PI3K) complex, which mediates vesicle trafficking. Autophagy is a self-degradation process that is important for balancing energy sources during critical periods during development and in response to cellular stress. Although autophagy is not a type of cell death, it is important for cartilage homeostasis. Generally speaking, autophagy promotes cell survival by enabling cells to adapt to stress conditions, but the process is also considered a non-apoptotic cell death program (Almonte-Becerril M, Navarro-Garcia F, Gonzalez-Robles A, Vega-Lopez MA, Lavalle C, Kouri JB. Cell death of chondrocytes is a combination between apoptosis and autophagy during the pathogenesis of Osteoarthritis within an experimental model. Apoptosis. 2010 May; 15(5): 631-8. doi: 10.1007 / s10495-010-0458-z. PMID: 20091349).

[0134] like Figure 7d As shown, the reduction of BECN-1 expression by etoposide and OSM was significantly offset by APPA 10ug / ml and PA 7.7ug / ml, indicating an effect on autophagy. Interestingly, APPA alone reduced BECN-1 expression levels to below that of unstimulated chondrocytes (p=0.051); see Figure 7d , column 8. Figure 7eFigure 2 shows the effects of APPA, AP, and PA on BCL2L13 gene expression. BCL2L13 encodes a mitochondrially localized protein with a conserved B-cell lymphoma 2 homology motif. Overexpression of the encoded protein leads to apoptosis. In human osteoarthritis tissue samples, apoptosis has been positively correlated with the severity of cartilage destruction and matrix depletion (doi:10.3390 / ijms16036093). The anti-apoptotic proteins Bcl-2 and Bcl-XL inhibit cytochrome c (cyt-c) release. (Musumeci G, Castrogiovanni P, Trovato FM, Weinberg AM, Al-Wasiyah MK, Alqahtani MH, Mobasheri A. Biomarkers of Chondrocyte Apoptosis and Autophagy in Osteoarthritis. Int J Mol Sci. 2015 Aug 31; 16(9):20560-75. doi:10.3390 / ijms160920560. PMID:26334269; PMCID:PMC4613218.).

[0135] APPA does not appear to regulate the expression of BCL2L13. This is consistent with the apoptosis results in Figure 4, which show that APPA does not have any obvious effect on either early or late apoptosis, and therefore the reduction in senescence caused by APPA administration is not caused by increased apoptosis.

[0136] Example 3 - Liquid Formulation

[0137] To produce a 1000 mg APPA formulation that is a stable liquid at room temperature, 777.8 mg of paeonol (obtained from Sigma-Aldrich Gillingham) was mixed with 222.2 mg of vanillyl acetone (obtained from Sigma-Aldrich Gillingham). The mixture was heated and 555.6 mg of PEG 400 was added while stirring the mixture. A stable mixture was produced that did not solidify when stored overnight at approximately 3°C.

[0138] Example 4 - Capsule Formulation - 400 mg APPA Capsules

[0139] To produce a formulation containing approximately 400 mg of APPA, 311.1 mg of paeonol (obtained from Sigma-Aldrich Gillingham) was mixed with 88.9 mg of vanillyl acetone (obtained from Sigma-Aldrich Gillingham). The mixture was heated and 222.2 mg of PEG 400 (obtained from Sigma-Aldrich Gillingham) was added while stirring the mixture. This produced 0.533 ml of a liquid formulation of paeonol and vanillyl acetone that was stable at room temperature. This can be encapsulated in a soft gelatin capsule by methods known in the art to provide a pharmaceutical product in capsule form.

[0140] Example 5 - Solid Formulation

[0141] To produce a solid dosage form, the following preparation can be used to make "O" shaped capsules (474.5 mg total).

[0142] Example 5 Unit mg / capsule Target Paeonol (synthetic) 311.1mg active Vanilla acetone (synthetic) 88.9mg active Excipient: Colloidal anhydrous silica (silicon dioxide) 0.5mg lubricant Excipient: Microcrystalline cellulose 70mg lubricant Excipient: magnesium stearate 4mg lubricant

[0143] Disclosed above are compositions and uses defined by the following numbered statements:

[0144] 1. A composition comprising vanillone or an isomer thereof and paeonol or an isomer thereof, for use in treating aging, wherein administration of the composition results in a decrease in the number of senescent cells and a simultaneous increase in the number of total viable cells.

[0145] 2. A composition comprising vanillone or an isomer thereof and paeonol or an isomer thereof, and the composition is used to treat aging through the mechanism of aging phenotype regulation.

[0146] 3. The composition of paragraph 1 or 2, wherein administration of the composition results in an increase in cell number.

[0147] 4. A composition comprising vanillone or an isomer thereof and paeonol or an isomer thereof, for treating aging through a senescent cell clearance mechanism.

[0148] 5. A composition according to any preceding paragraph comprising vanillone and paeonol.

[0149] 6. A composition according to any preceding paragraph wherein the ratio of paeonol to vanillone (by weight) is from 3:2 to 9:1.

[0150] 7. A composition according to any preceding paragraph, wherein the formulation is a liquid formulation.

[0151] 8. The composition according to any one of paragraphs 1 to 6, wherein the formulation is a solid formulation, such as a pill, tablet or capsule.

[0152] 9. A composition according to any preceding paragraph for use in treating an age-related disease.

[0153] 10. A composition according to any preceding paragraph for use in treating osteoporosis.

[0154] 11. A composition according to any preceding paragraph for use in treating sarcopenia.

[0155] 12. A composition according to any preceding paragraph for use in treating chronic obstructive pulmonary disease.

[0156] 13. A composition according to any preceding paragraph for use in the treatment of idiopathic pulmonary fibrosis.

[0157] 14. A composition according to any preceding paragraph for use in treating a lung disease, such as pulmonary hypertension and pulmonary arterial hypertension.

[0158] 15. A composition according to any preceding paragraph for use in the treatment of hepatic steatosis.

[0159] 16. A composition according to any preceding paragraph for use in the treatment of kidney diseases such as acute kidney injury (AKI), chronic kidney disease (CKD), tubulointerstitial fibrosis (TIF), glomerulosclerosis and vascular sclerosis.

[0160] 17. A composition according to any preceding paragraph for use in treating atherosclerosis.

[0161] 18. A composition according to any preceding paragraph for use in the treatment of type 1 diabetes.

[0162] 19. A composition according to any preceding paragraph for use in treating age-related macular degeneration.

[0163] 20. A composition according to any preceding paragraph for use in the treatment of Alzheimer's disease, Parkinson's disease, frontotemporal dementia, amyotrophic lateral sclerosis and / or multiple sclerosis.

[0164] 21. A composition according to any preceding paragraph, wherein the composition is administered orally.

[0165] 22. Use of vanillone or its isomers and paeonol or its isomers in the preparation of a medicament for treating aging.

[0166] 23. Use of vanillone or its isomers and paeonol or its isomers in the preparation of a medicament for treating age-related diseases.

[0167] 24. Use of vanilla acetone or its isomers and paeonol or its isomers in the preparation of a medicament for treating osteoporosis, sarcopenia, lung diseases such as chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), pulmonary hypertension (PH) and pulmonary arterial hypertension (PAH), liver diseases such as acute liver injury, chronic liver disease and hepatic steatosis, kidney diseases such as acute kidney injury (AKI), chronic kidney disease (CKD), tubulointerstitial fibrosis (TIF), glomerulosclerosis and vascular sclerosis, vascular diseases such as atherosclerosis, type 1 diabetes, age-related macular degeneration (AMD), Alzheimer's disease (AD), Parkinson's disease, frontotemporal dementia, amyotrophic lateral sclerosis or multiple sclerosis.

[0168] 25. A method for treating age-related diseases, comprising the steps of: administering a composition comprising vanillone or an isomer thereof and paeonol or an isomer thereof to a patient in need thereof; or

[0169] A method for treating aging, comprising the steps of: administering a composition comprising vanillone or an isomer thereof and paeonol or an isomer thereof to a patient in need thereof; or

[0170] A method of treating osteoporosis, sarcopenia, lung diseases such as chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), pulmonary hypertension (PH) and pulmonary arterial hypertension (PAH), liver diseases such as acute liver injury, chronic liver disease and hepatic steatosis, kidney diseases such as acute kidney injury (AKI), chronic kidney disease (CKD), tubulointerstitial fibrosis (TIF), glomerulosclerosis and vascular sclerosis, vascular diseases such as atherosclerosis, type 1 diabetes, age-related macular degeneration (AMD), Alzheimer's disease (AD), Parkinson's disease, frontotemporal dementia, amyotrophic lateral sclerosis or multiple sclerosis;

[0171] The method comprises the steps of administering a composition comprising vanillone or an isomer thereof and paeonol or an isomer thereof to a patient in need thereof.

Claims

1. A composition comprising vanillone or an isomer thereof and paeonol or an isomer thereof, for use in treating aging, wherein administration of the composition results in a decrease in the number of senescent cells and a simultaneous increase in the number of total viable cells.

2. A composition comprising vanillone or an isomer thereof and paeonol or an isomer thereof, and the composition is used to treat aging through the mechanism of aging phenotype regulation.

3. A composition comprising vanillone or an isomer thereof and paeonol or an isomer thereof, for treating aging through a senescent cell clearance mechanism.

4. A composition according to any preceding claim comprising vanillone and paeonol.

5. A composition according to any preceding claim, wherein the ratio of paeonol to vanillone (by weight) is from 3:2 to 9:

1.

6. A composition according to any preceding claim, wherein the formulation is a liquid formulation.

7. The composition according to any one of claims 1 to 6, wherein the preparation is a solid preparation, such as a pill, tablet or capsule.

8. A composition according to any preceding claim for use in the treatment of an age-related disease.

9. A composition according to any preceding claim for use in the treatment of osteoporosis.

10. A composition according to any preceding claim for use in the treatment of sarcopenia.

11. A composition according to any preceding claim for use in the treatment of chronic obstructive pulmonary disease.

12. A composition according to any preceding claim for use in the treatment of idiopathic pulmonary fibrosis.

13. A composition according to any preceding claim for use in the treatment of a lung disease, such as pulmonary hypertension and pulmonary arterial hypertension.

14. A composition according to any preceding claim for use in the treatment of hepatic steatosis.

15. A composition according to any preceding claim for use in the treatment of kidney diseases such as acute kidney injury (AKI), chronic kidney disease (CKD), tubulointerstitial fibrosis (TIF), glomerulosclerosis and vascular sclerosis.

16. A composition according to any preceding claim for use in the treatment of atherosclerosis.

17. A composition according to any preceding claim for use in the treatment of type 1 diabetes.

18. A composition according to any preceding claim for use in the treatment of age-related macular degeneration.

19. A composition according to any preceding claim for use in the treatment of Alzheimer's disease, Parkinson's disease, frontotemporal dementia, amyotrophic lateral sclerosis and / or multiple sclerosis.

20. A composition according to any preceding claim, wherein the composition is administered orally.

21. Use of vanillone or its isomers and paeonol or its isomers in the preparation of a medicament for treating aging.

22. Use of vanillone or its isomers and paeonol or its isomers in the preparation of a medicament for treating age-related diseases.

23. Use of vanilla acetone or its isomers and paeonol or its isomers in the preparation of a medicament for treating osteoporosis, sarcopenia, lung diseases such as chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), pulmonary hypertension (PH) and pulmonary arterial hypertension (PAH), liver diseases such as acute liver injury, chronic liver disease and hepatic steatosis, kidney diseases such as acute kidney injury (AKI), chronic kidney disease (CKD), tubulointerstitial fibrosis (TIF), glomerulosclerosis and vascular sclerosis, vascular diseases such as atherosclerosis, type 1 diabetes, age-related macular degeneration (AMD), Alzheimer's disease (AD), Parkinson's disease, frontotemporal dementia, amyotrophic lateral sclerosis, or multiple sclerosis.

24. A method for treating age-related diseases, comprising the steps of: administering to a patient in need thereof a composition comprising vanillone or an isomer thereof and paeonol or an isomer thereof; or A method for treating aging, comprising the following steps: administering to a patient in need thereof a composition comprising vanillone or an isomer thereof and paeonol or an isomer thereof; or A method of treating osteoporosis, sarcopenia, lung diseases such as chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), pulmonary hypertension (PH) and pulmonary arterial hypertension (PAH), liver diseases such as acute liver injury, chronic liver disease and hepatic steatosis, kidney diseases such as acute kidney injury (AKI), chronic kidney disease (CKD), tubulointerstitial fibrosis (TIF), glomerulosclerosis and vascular sclerosis, vascular diseases such as atherosclerosis, type 1 diabetes, age-related macular degeneration (AMD), Alzheimer's disease (AD), Parkinson's disease, frontotemporal dementia, amyotrophic lateral sclerosis, or multiple sclerosis; The method comprises the following steps: A composition comprising vanillone or an isomer thereof and paeonol or an isomer thereof is administered to a patient in need thereof.

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